The research described in this proposal is designed to elucidate the mechanisms by which phospholipids are assembled into mitochondrial and plasma membranes; and to determine the role that specific phospholipids play in the structure and function of these membranes. These goals will be accomplished by establishing mutants of Chinese hamster ovary (CHO) cells that are deficient in the synthesis of phosphatidylethanolamine and sphingomyelin. Under conditions of ethanolamine starvation the synthesis of phosphatidylethanolamine in CHO cells is dependent upon the intracellular transport of phosphatidylserine from the endoplasmic reticulum to the mitochondrion. Thus, by selecting for ethanolamine auxotrophs in CHO cells it will be possible to identify a subclass of mutants that are incapable of the intracellular transport of phosphatidylserine to the mitochondria. By using the techniques of in vitro complementation between mutant and parental cells we will identify the factors responsible for this transport process, and determine whether they are related to the phospholipid exchange/transfer proteins. Ethanolamine auxotrophs will also be used to examine the effects of phosphatidylethanolamine depletion upon membrane structure and function. Sphingomyelin synthesis is dependent upon the intracellular transport of phosphatidylcholine and ceramide from the endoplasmic reticulum to the plasma membrane. By selecting mutants of CHO cells that either require sphingomyelin for growth, or are unable to express sphingomyelin as a surface antigen, it will be possible to identify mutants that are deficient in the intracellular transport of ceramide. These mutants will be used to critically evaluate the role of phospholipid exchange/transfer proteins in the assembly of plasma membranes. These mutants will also be used to determine the effects of sphingomyelin depletion upon plasma membrane structure and function. Full biochemical characterization of these mutants will provide important insights into the mechanisms of membrane biogenesis in mammalian cells. The regulation of these mechanisms must ultimately be related to control of cell growth and differentiation.